Food Chemistry 138 (2013) 16961705

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Food Chemistry
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Relationships between harvest time and wine composition in Vitis vinifera L.

cv. Cabernet Sauvignon 1. Grape and wine chemistry
Keren Bindon a,, Cristian Varela a, James Kennedy a,b, Helen Holt a, Markus Herderich a
a
b

The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, South Australia 5064, Australia
California State University Fresno, Department of Viticulture & Enology, 2360 E. Barstow Avenue MS VR89, Fresno, CA 93740-8003, USA

a r t i c l e

i n f o

Article history:
Received 14 August 2012
Received in revised form 28 September 2012
Accepted 28 September 2012
Available online 10 November 2012
Keywords:
Grape
Ripeness
Wine
Methoxypyrazine
C6 alcohols
Ester
Volatile
Aroma
Tannin
Phenolic
Polysaccharide

a b s t r a c t
The study aimed to quantify the effects of grape maturity on wine alcohol, phenolics, avour compounds
and polysaccharides in Vitis vinifera L. cv Cabernet Sauvignon. Grapes were harvested at juice soluble solids from 20 to 26 Brix which corresponded to a range of wine ethanol concentrations between 12% and
15.5%. Grape anthocyanin and skin tannin concentration increased as ripening progressed, while seed
tannin declined. In the corresponding wines, monomeric anthocyanin and wine tannin concentration
increased with harvest date, consistent with an enhanced extraction of skin-derived phenolics. In wines,
there was an observed increase in yeast-derived metabolites, including volatile esters, dimethyl sulde,
glycerol and mannoproteins with harvest date. Wine volatiles which were signicantly inuenced by harvest date were isobutyl methoxypyrazine, C6 alcohols and hexyl acetate, all of which decreased as ripening progressed. The implications of harvest date for wine composition is discussed in terms of both grape
composition and yeast metabolism.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
During grape ripening, multiple biochemical processes occur at
different rates, and are stage-specic (Zamboni et al., 2010), consequently the chemical composition of grapes destined for winemaking can be dened temporally. For grape-derived compounds
which may positively or negatively inuence wine chemistry and
sensory properties, these may be in a state of increasing, decreasing or remaining constant at a given point in grape development.
Hence, decision-making in terms of harvest date for commercial
winemaking requires the consideration of complex factors. A harvesting decision for optimal ripeness requires adequate knowledge of grape compositional factors relevant to achieve a
targeted wine style, taking into consideration the grape cultivar,
climate, topography, seasonal weather conditions and vineyard
management practices. Traditional measures used to determine
grape ripeness include the assay of juice total soluble solids as an
estimate of grape sugar accumulation, or an estimate of grape acidity decline as titratable acids or pH (Jackson & Lombard, 1992).
However, there is general recognition that these measures alone
are not sufcient to accurately predict wine composition, notably
as many key grape-derived compounds do not track with sugar
Corresponding author. Tel.: +61 8 83136190; fax: +61 8 83136601.
E-mail address: keren.bindon@awri.com.au (K. Bindon).
0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.09.146

accumulation, and are also highly dependent upon the grapevine

genotype and its environment (Jackson & Lombard, 1992).
In terms of grape cultivars, the variety Vitis vinifera L. cv. Cabernet Sauvignon presents an interesting case study due to a frequently observed dichotomy of sensory attributes, distinguishing
vegetative from fruity characters (Robinson et al., 2011). Advances in avour chemistry have shed light on this commercially
important grape variety, dening potential avour precursors in
grapes and predicting their sensorial impact in wine (Forde, Cox,
Williams, & Boss, 2011; Kalua & Boss, 2009; Roujou de Boube,
Van Leeuwen, & Dubourdieu, 2000). While vegetative characteristics in Cabernet Sauvignon have long been thought to be due to the
presence of isobutyl methoxypyrazine (IBMP) which denotes a
green bell pepper attribute (Rojou de Boube et al., 2000; Cacho
& Ferreira, 2010; Robinson et al., 2011), the C6 alcohols and their
derivatives have also been implicated in this sensory attribute as
herbaceous or green (Escudero, Campo, Farina, Cacho, & Ferreira,
2007; Kalua & Boss, 2009). IBMP is known to consistently decrease
during grape ripening, which has potential implications for vegetative aroma in wines according to harvest date (Rojou de Boube
et al., 2000). Grape C6 derivatives in situ can exist as acetate esters,
aldehydes or alcohols, with C6-alcohols predominating during the
later stages of grape ripening (Kalua & Boss, 2009). However, in
grape crushing and during fermentation there is the potential de

K. Bindon et al. / Food Chemistry 138 (2013) 16961705

novo generation of additional C6 derivatives from fatty acid precursors via the lipoxygenase pathway. The conversion of C6
derivatives to acetates by yeast activity, may alter the expected
herbaceous prole, in particular since hexyl-acetate may be associated with fruity attributes (Dennis et al., 2012; Forde et al., 2011).
While impact odourants are important in conferring wine sensory attributes, Escudero et al. (2007) and Cacho (2010) have
emphasised that synergistic interactions between wine volatiles
can lead to the enhancement or dampening effects on aroma/
avour attributes. For example, in terms of wine fruitiness,
yeast-derived esters which are largely responsible for this attribute
can either mask vegetative odours, or ester aroma may be enhanced by the presence of norisoprenoids or dimethyl sulde in
low concentrations (Escudero et al., 2007). In terms of assessing
the effects of grape ripening on wine volatiles, it is therefore also
important to recognise the signicant role of yeast metabolites.
For Cabernet Sauvignon, astringency has been found to be a signicant factor which discriminates wine palate, or taste (Robinson
et al., 2011). Interestingly, this has been found to be negatively correlated with vegetative aroma, and strongly associated with the
concentration of condensed tannin (proanthocyanidin) in wines
(Robinson et al., 2011). In red grape varieties like Cabernet Sauvignon, the assessment of compositional changes in grape phenolics
during ripening, denoted phenolic ripeness has been emphasised
as an important consideration (Jackson & Lombard, 1992; PerezMagarino & Gonzalez-San Jose, 2006). To date, a clear relationship
between the tannin concentration of grapes, and the resulting tannin concentration in wines has not been demonstrated, in particular with respect to grape ripeness (Fournand et al., 2006). Work by
Ristic, Bindon, Francis, Herderich, and Iland (2010) has demonstrated a strong relationship between grape skin tannin concentration and wine tannin concentration, while seed tannin showed a
poor relationship. This nding, and the observation that a higher
tannin concentrations together with a higher proportion of skin
tannin are correlated with increases in commercial wine allocation
grading (Kassara & Kennedy, 2011), highlights the need for a greater understanding of tannin partitioning from grape solids to wine
as it relates to grape ripening. Further, accumulation of the coloured pigments anthocyanins occurs following the onset of ripening (veraison), and increased grape colour has been correlated with
enhanced wine colour and ageing capacity (Perez-Magarino &
Gonzalez-San Jose, 2006). Anthocyanins have the capacity to bind
to tannin under the conditions of vinication (Hayasaka & Kennedy, 2003), and changes in this class of phenolics therefore has
signicant implications for both wine astringency, and colour stability as bisulphite-resistant (polymeric) pigments.
The current study provides a holistic overview of key components of the variety Cabernet Sauvignon, tracking composition
from grape to wine. While limited to a single season, we have
aimed to present a case study whereby wine composition can be
inferred from the temporal changes in grape composition during
ripening. This study has sought to incorporate the impact of both
grape biochemistry and yeast metabolism on wine composition
in order to account for differences in classes of compounds.
2. Materials and methods
2.1. Instrumentation
For HPLC analyses an Agilent model 1100 HPLC (Agilent Technologies Australia Pty. Ltd., Melbourne, Australia) was used. For
analysis of grape and wine volatiles an Agilent gas chromatograph
(GC) (6890 series) coupled with an Agilent 5973/5973N/5975A
mass selective detector (MS) (Agilent Technologies Australia Pty.
Ltd., Melbourne, Australia) was used. For the analysis of monosaccharides as their alditol acetates a HewlettPackard GC (6890 ser-

1697

ies) coupled with a HewlettPackard 5973 MS (HewlettPackard

Australia, North Ryde, NSW, Australia) (GCMS) was used. Methods used Agilent Chemstation software for data analysis (Agilent
Technologies Australia Pty. Ltd., Melbourne, Australia).
2.2. Harvesting, grape sampling and preparation for analysis
Vitis vinifera L. cv. Cabernet Sauvignon samples were obtained
from a commercial vineyard in the Langhorne Creek growing region
of South Australia, Australia which has a latitude 35 1611.5600 S and
a longitude 139 0014.4700 E, with an elevation approximately 28 m
above sea level. The grapevines were 12 years old, planted 2.5 m
(row)  1.8 m (vine); on own roots, spur pruned, with a single-wire
trellis and sprawling canopies. Irrigation for the 2009/2010 growing
season was 272 mm (0.5 ML/ha). The average yield was 6 kg/vine,
and winter pruning weight for 2010 was 0.9 kg/vine, giving a pruning:yield ratio (Ravaz index) of 6.7. Grape samples were obtained
at ve different stages of ripeness in the 2009/2010 growing season.
Harvest dates were on the 16th and 23rd February, and on the 2nd,
10th and 17th of March, and are designated as sequential treatments
H1 to H5 in this study. To obtain a representative sample, approximately 3  70 kg of grapes were harvested from the same three rows
distributed within the vineyard block. The rows had approximately
100 grapevines/row. For each harvest date, grapevines were selected
at staggered points across each row, and completely harvested. In order to minimise within-vineyard differences in total soluble solids
(TSS), the grape lots were well mixed, and sub-divided into
3  50 kg lots for small-scale winemaking. Three replicate 100-berry
samples of each harvest were processed fresh, and manually separated into juice, skin and seed components, while kept on ice. Skins
and seeds were then frozen in liquid nitrogen and stored at 80 C
until processed. A further 2  200-berry samples were collected for
each winemaking replicate. One was frozen at 20 C, and the second was pressed by hand in a small plastic bag to express the juice.
Fresh juice from the 100- and 200-berry samples and was then centrifuged at 1730g for 5 min. Juice from the 100-berry sample was frozen at 20 C until processed. The fresh juice from the 200-berry
sample was directly analysed.
2.3. Small-scale winemaking
Three 50 kg replicate lots of grapes harvested at different ripeness stages were crushed and de-stemmed with the addition of
50 ppm SO2. Based on the minimum pH of the rst harvest date
of 3.2, musts from subsequent dates were adjusted to approximate
this pH using tartaric acid. Based on the measured YAN content of
each must, total assimilable nitrogen was adjusted to 250 mg/L
with diammonium phosphate (DAP). The yeast used was S. cerevisiae PDM (Maurivin, Sydney, Australia) which is analogous to
EC1118 (Novo et al., 2009). Yeast was inoculated at 200 mg/kg,
and fermentation was carried out on the skins for 7 days in a temperature-controlled room at 15 C, and plunged 20 times twice daily. Thereafter, the ferments were drained and pressed and the
combined free-run juice and pressed wine fermented to dryness.
Wines were then racked off gross lees, and 60 ppm SO2 added,
and were acid-adjusted to pH 3.5 with tartaric acid. Wines were
then cold-stabilised at 0 C for a minimum of 21 days, and thereafter racked off ning lees. The nal SO2 level was adjusted to a total
of 80 ppm (free 40 ppm), since no malolactic fermentation was
performed. Wines were ltered through a 0.8 lm membrane and
bottled in 750 mL bottles under screw-cap.
2.4. General analysis of fresh juice and wine
Juice TSS as Brix was determined using a digital refractometer.
Juice and wine pH and titratable acidity (TA) were determined

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K. Bindon et al. / Food Chemistry 138 (2013) 16961705

using a pH meter and combination electrode. The TA was

determined by titrating with 0.33 M sodium hydroxide solution
to a pH end-point of 7. The TA is expressed in g/L of tartaric acid
equivalents. Ammonia concentration in fresh juices was determined using the Glutamate Dehydrogenase Enzymatic Bioanalysis
UV-method test (Roche, Mannheim, Germany). Juice free a-amino
acid nitrogen (FAN) was determined by the o-phtaldehyde/N-acetyl-L-cysteine spectrophotometric assay procedure, which excludes
proline (Dukes & Butzke, 1998). Juice yeast-assimilable nitrogen
(YAN) was calculated by adding the nitrogen present in ammonium
to the FAN concentration. Wine ethanol concentration was determined using a Foss WineScan FT 120 as described by the manufacturer (Foss, Hillerd, Denmark). The concentrations of residual
sugar, glycerol, malic acid, succinic acid and acetic acid were measured by high-performance liquid chromatography (HPLC) using a
Bio-Rad HPX-87H column as described previously (Varela, Pizarro,
& Agosin, 2004).

2.5. Analysis of methoxypyrazines in grape juice and wine

A frozen (20 C) 200-berry sample was homogenised at
8000 rpm for 2  20 s in a Retsch Grindomix GM200 (Retsch GmbH
& Co, Haan, Germany). A 40 g sub-sample of the homogenate was
centrifuged to obtain claried juice. A 20 mL aliquot of either juice
or wine was taken and extracted for analysis of methoxypyrazines,
to which a 50 lL aliquot of deuterated isobutyl methoxypyrazine
(IBMP-d3) (CDN Isotopes, SciVac Pty. Ltd., Hornsby, NSW, Australia)
was added in a 50 mL centrifuge tube and gently mixed. The solution was adjusted to a pH < 1 using a sulphuric acid solution and
centrifuged at 3200 g for 3 min. The acidied solution was eluted
through a pre-conditioned cation-exchange (Varian Bond Elut
SCX) solid-phase extraction cartridge (Agilent Technologies
Australia Pty. Ltd., Melbourne, Australia). The cartridge was
washed with two volumes of Milli-Q water and air-dried under
vacuum. The cartridge was eluted with 2% (w/v) NaOH and collected in a 20 mL solid-phase micro-extraction (SPME) vial. Sodium
chloride (2 g) was added and the vial capped and analysed using
GC/MS with headspace SPME equipped with a DB-5MS (30 m 
0.25 mm  0.25 lm) capillary column (Agilent Technologies
Australia Pty. Ltd., Melbourne, Australia) and a standard splitsplitless injector. The SPME bre was a J&W DVB/Carboxen/PDMS
(StableFlex, Agilent, Australia) which was preconditioned in the
GC injector at 270 C for 60 min. Samples were equilibrated
5 min at 50 C prior to SPME adsorption at 45 min at 50 C, with
periodic agitation. The SPME desorption time was 600 s with the
inlet temperature at 250 C. The initial oven temperature was
50 C held for 5 min, and the oven ramp program was for 5 C/
min until 100 C, followed by 25 C/min increments until 250 C
which was held for 10 min. The column ow rate was constant
at 1.5 mL/min, with helium as the carrier gas. IBMP, isopropyl
methoxypyrazine (IPMP) and sec-butyl methoxypyrazine (SBMP)
were quantied using a multi-level set of matrix-matched standards (Sigma Aldrich, St. Louis, MO, USA; Pyrazine Specialties,
Atlanta, GA, USA) and IBMP-d3 as a surrogate standard. The MS
was operated in EI mode with a multiplier gain of 400 mV and in
selective ion monitoring (SIM) mode. Target ions and qualier ions
(in parenthesis) were m/z 154 (127/94) for IBMP-d3, m/z 124 (94/
151) for IBMP, m/z 152 (124/137) for IPMP, and m/z 138 (124/
151) for SBMP. Linearity for the concentration range 5200 ng/L
was >0.99, the limit of detection was 5 ng/L, and the coefcient
of variation was <10% for all methoxypyrazines assayed at both
5 ng/L and 100 ng/L. The uncertainty of measurement was estimated at 20% [2  the coefcient of variation] or an absolute
amount of 2 ng/L, whichever was the greater.

2.6. General analysis of wine volatiles

Grape-derived volatile compounds, norisorpenoids (rose oxide,
b-damascenone and b-ionone) and monoterpenes (linalool, a-terpineol, nerol and geraniol) were analysed using headspace SPME
coupled with GCMS (HS/SPME-GC/MS), with polydeuterated internal standards for stable isotope dilution analysis (SIDA) as described elsewhere (Pedersen, Capone, Skouroumounis, Pollnitz, &
Sefton, 2003; Ugliano, Siebert, Mercurio, Capone, & Henschke,
2008). C6 compounds (hexanol, E-2-hexenal, Z-3-hexen-1-ol and
E-2-hexen-1-ol) were also analysed by HS-SPME/GCMS using hexanol-d13, E-2-hexenal-d9 and E-2-hexen-1-ol-d4 as internal standards as previously described (Capone, Black, & Jeffery, 2012).
Yeast-derived sulphur-containing volatiles (carbon disulde,
diethyl disulde, dimethyl sulde, ethanethiol, ethyl thioacetate,
hydrogen sulde, methanethiol and methyl thioacetate) were
determined by using headspace cool-on-column gas chromatography coupled with an atomic emission detector (HS-COC-GCAED),
with ethylmethyl sulde and propyl thioacetate as internal standards (Siebert, Solomon, Pollnitz, & Jeffery, 2010). Yeast volatile
fermentation products were analysed using HS-SPMEGCMS, with
SIDA as described previously (Siebert et al., 2005). Fifteen compounds, including ethyl- and acetate esters and higher alcohols,
were quantied.
2.7. Grape and wine colour and tannin analysis
For grape tannin extraction, frozen skins and seeds were
extracted in 70% v/v acetone in water for 18 h. An aliquot of the
extract was dried under a stream of nitrogen and reconstituted
in 50% v/v ethanol in water. Grape tannin extracts and wines were
analysed by the methyl cellulose precipitable tannin (MCPT) assay
using the high-throughput method described in (Mercurio, Dambergs, Herderich, & Smith, 2007). For analysis of total grape anthocyanin, a frozen (20 C) 200-berry sample was homogenised at
8000 rpm for 2  20 s in a Retsch Grindomix GM200 (Retsch GmbH
& Co., Haan, Germany). A 1 g sub-sample of homogenate was
extracted according to (Mercurio et al., 2007) and anthocyanin
concentration and content determined using the original method
outlined in (Iland, Bruer, Edwards, Weeks, & Wilkes, 2004). Wine
total anthocyanin and colour parameters were determined according to (Mercurio et al., 2007). Wine tannin was analysed using
phloroglucinolysis and gel permeation chromatography following
isolation by solid-phase extraction according to the methods
described in (Kassara & Kennedy, 2011). Pre-veraison skin tannin
fractions of known mean degree of polymerisation (mDP) (by
phloroglucinolysis) were used as standards for calibration. For
GPC calibration, a second order polynomial was tted with the
tannin elution time at 50% for each standard. For the MCPT assay,
phloroglucinolysis and GPC analysis, ()-epicatechin (Sigma
Aldrich, St. Louis, MO, USA) was used as the quantitative standard.
2.8. Grape and wine polysaccharide analysis
Grape juices and wines were concentrated 4 times. Frozen
grape juice (20 C) was concentrated by lyophilisation in order
to prevent degradation by endogenous enzymes, and wine was
concentrated under a stream of nitrogen at 30 C. Thereafter, grape
and wine soluble polysaccharides were isolated from each experimental replicate through precipitation in ethanol as described previously (Ayestarn, Guadalupe, & Len, 2004; Vidal, Williams,
Doco, Moutounet, & Pellerin, 2003). Neutral monosaccharides were
analysed following hydrolysis in 2 M triuoroacetic acid for 90 min
at 100 C. Hydrolysates were concentrated under a stream of
nitrogen, and then analysed either as their alditol acetates by
GCMS on a BPX70 capillary column (SGE, Ringwood, Victoria,

K. Bindon et al. / Food Chemistry 138 (2013) 16961705

Australia) (Lau & Bacic, 1993; Sims & Bacic, 1995) or by HPLC as
their 1-phenyl-3-methyl-5-pyrazolone derivatives (Honda et al.,
1989). The uronic acid content was determined colourimetrically
using D-galacturonic acid (Sigma Aldrich, St. Louis, MO, USA) as
the quantitative standard (Filisetti-Cozzi & Carpita, 1991). Polysaccharides were analysed by size exclusion chromatography (SEC)
using the method of Vidal et al. (2003) with the following modications. Dry samples were resuspended in 0.1 M sodium nitrate
and 25 lL was injected onto a BioSep Sec 2000 column
(300  7.8 mm, Phenomenex, Lane Cove, NSW, Australia) and analysed by HPLCSEC in 0.1 M sodium nitrate at a ow rate of 1 mL/
min. Polysaccharide elution was monitored by refractive index
detection over 14 min. Column calibration was carried out with
commercial dextrans having molecular weights ranging from 5 to
270 kDa (Sigma Aldrich, St. Louis, MO, USA). In order to conrm
the elution prole of the HPLCSEC method, polysaccharide isolates were fractionated by semi-preparative SEC on a glass column
(Amersham Biosciences, Uppsala, Sweden) packed with Sephacryl
S-300 gel (Pharmacia, Uppsala, Sweden) of bed volume 400 cm3
using 0.1 M sodium nitrate at a ow rate of 1 mL/min. Fractions
eluting at successive time points were collected, dialysed (7 kDa
cut-off) against Milli-Q water for 48 h, and lyophilised. Polysaccharide fractions were identied as mannoproteins (MPs), arabinogalactan proteins (AGPs) or rhamnogalacturonans (RGs) based on
their relative monosaccharide composition and by comparison
with published data (Vidal et al., 2003). Separation by HPLCSEC
was incomplete, but elution ranges for the different polysaccharide
classes were as follows: MPs = 5.88 min (185 kDa); AGPs 6.3
8.0 min (2448 kDa); RGs = 8.18.7 min (5.8 kDa); low molecular
weight polysaccharides 10 min (<5.8 kDa).
2.9. Statistical analysis
Signicant differences between harvest dates were determined
from triplicate experiments using a one-way analysis of variance
(ANOVA), followed by a post hoc Students t-test. The JMP 5.0.1 statistical software package (SAS, Cary, NC, USA) was used.
3. Results and discussion
3.1. Grape berry sugar accumulation
Over the ve sampling stages, grape soluble solids, expressed as
Brix of sugar increased continually from 20.3 (H1) to 26.0 (H5)
(Table 1). The analysis of grape juice sugars as total soluble solids
in order to track ripening and estimate harvest time and nal wine
alcohol is a traditional practice in wine production (Iland et al.,
2004). Alternately, grape sugar can be expressed as sugar per berry
in order to provide information on sugar loading (Deloire, 2011).
This manner of monitoring the ripening process provides information on grapevine physiology, and allows grape berry sugar accumulation kinetics to be inferred. Typically, the ripening process is
characterised by an initial active sugar accumulation phase followed by a plateau, notwithstanding that total soluble solids in
juice may increase due to a reduction in berry size in the later
stages of ripening (Bindon, Dry, & Loveys, 2008; Deloire, 2011). A
divergence from this model has been suggested to be due to an
imbalance in source:sink relationship within the grapevine (Deloire, 2011). For the site used in this study, the calculated Ravaz index of 6.7 suggests that the grapevines were not unbalanced
(Bravdo, Hepner, Loinger, Cohen, & Tabacman, 1985) and it has
been noted previously that the site was irrigated.
The rate of sugar accumulation per berry (mg/berry/day)
(Table 1) was determined using historical data (Bindon, Bacic, &
Kennedy 2012) and revealed that sugar accumulation was initially

1699

>3 mg/berry/day for H1 and H2, and slowed by H3 and H4. According to the model developed for grapevines (Deloire, 2011) this
would indicate a plateau had been reached when sugar loading is
<3 mg/berry/day. However, an unexpected increase in sugar loading was observed from H4 to H5, which was noted to follow a rain
event and was a period associated with moderate temperatures
(Supplementary data S1). It has been noted that water decit reduces photosynthesis, and as a result impedes sugar loading during
the late stages of grape development (Wang, Deloire, Carbonneau,
Federspiel, & Lopez, 2003). The later accumulation in sugar may
therefore have resulted from a transient enhancement in photosynthesis following the rain event, and we suggest was not the result of an imbalance in carbohydrate partitioning within the
grapevines.
3.2. Grape composition
Grape juice TA decreased during the ripening period and was
associated with an increase in pH, and pH did not exceed 3.5 by
the nal sampling date (Table 1). As discussed previously, YAN levels were low (maximum 106 mg/L), and decreased as ripening progressed. YAN decreases corresponded to drops in both a-amino
nitrogen and ammonia. The grape-derived aroma compound IBMP,
typical to Cabernet Sauvignon, decreased between H3 and the nal
two harvest points H4 and H5. The decrease in grape-derived IBMP
during ripening is expected for this variety (Rojou de Boube et al.,
2000). Grape juice soluble polysaccharides, as the sum of their
respective acidic monosaccharides (primarily galacturonans) and
neutral monosaccharides did not show a signicant trend during
ripening. The neutral monosaccharide fraction did not undergo
any compositional changes, and were on average 43% galactose
and 37% arabinose in molar proportion, with detectable but minor
contributions of glucose, rhamnose, xylose, fucose and mannose
(data not shown, Supplementary information S2). This analysis
indicates that the juice-soluble neutral polysaccharides were primarily arabinogalactan proteins.
The phenolic composition of the grape skin and seed components (solids) was determined in terms of total anthocyanin and
tannin (Table 1). Anthocyanin, expressed on a berry mass basis
(concentration) or per berry (content) increased throughout the
ripening period. Seed tannin decreased, and skin tannin increased
as ripening progressed, when expressed both as concentration
and content. This resulted in a progressive, and signicant increase
in the skin to seed tannin ratio, which was doubled between H1
and H5. Since the seed tannin concentration was higher than skin
tannin concentration, this resulted in a net decrease in total grape
tannin during ripening.
3.3. Wine ethanol content and non-volatiles
Ethanol concentration increased from 11.8% (v/v) at H1 to 15.5%
(v/v) at H5 (Table 2). To test consistency in the conversion of grape
sugar to ethanol, yeast fermentation efciency, dened as the concentration of sugar needed to produce 1% (v/v) of ethanol was calculated. This was found not to be inuenced by grape maturity and
was on average 16.85 g/L per 1% (v/v) regardless of initial sugar
concentration. Practically all sugar was consumed during fermentation, nevertheless residual sugar concentration was slightly higher for wines from H5. Yeast fermentation efciency remained
unaffected by grape maturity indicating that a set fraction of carbon is destined for ethanol production in the range of sugar concentrations evaluated in this study.
Wine pH and TA did not show any clear trend with grape maturity due to the acid adjustment employed as part of the vinication
process. The H4 treatment had lower TA, most likely due to low
tartaric acid concentration, which may reect a minor loss during

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K. Bindon et al. / Food Chemistry 138 (2013) 16961705

Table 1
General compositional analysis, non-volatile compounds and isobutyl methoxypyrazine in grape juice and solids from different harvest points in 2010 where H1 was the earliest
(16th February) and H5 the latest (17th March) sampling date.

Juice composition
Soluble sugar [Brix]f
Soluble sugar per berry [mg/berry]f
Rate of sugar accumulation [mg/berry/day]f
pH
Titratable acidity pH 7.0 [g/L]g
a-Amino nitrogen [mg/L]
Ammonia [mg/L]
Yeast-assimilable nitrogen [mg/L]
Neutral polysaccharide [mg/L]h
Acidic polysaccharide [mg/L]i
Total polysaccharide [mg/L]
Isobutyl methoxypyrazine [ng/L]
Grape solids composition
Anthocyanin [mg/g]j
Anthocyanin [mg/berry]j
Total tannin [mg/g]k
Total tannin [mg/berry]k
Seed tannin [mg/g]k
Seed tannin [mg/berry]k
Skin tannin [mg/g]k
Skin tannin [mg/berry]k
Skin tannin:seed tannin ratiol
EGCm of skin tannin extension subunits (% w/w)

H1

H2

H3

H4

H5

20.3 0.12a
199.5 1.1a
4.78
3.18 0.01a
8.3 0.2a
53 2.0a
57 0.9a
100 2.9a
149 11
328 31
477 29
8.3 0.3abc

22.1 0.12b
226.8 1.2b
3.79
3.18 0.02a
6.9 0.3b
57 4.6a
60 9.4a
106 12.2a
145 8.4
329 21
474 24
8.7 0.7ab

23.1 0.15c
229.4 1.5b
0.47
3.33 0.01b
6.5 0.05b
31 3.6bc
46 3.5ab
69 5.9b
135 9.0
247 21
382 16
9.0 1.2a

24.1 0.1d
239.6 0.9c
1.24
3.33 0.01b
5.7 0.21c
36 1.7b
31 1.0b
61 1.9b
125 13
287 49
412 39
6.3 0.3c

26.0 0.0e
280.5 1.3d
5.68
3.48 0.01c
5.3 0.25c
23 4.0c
33 3.9b
51 7.4b
150 22
273 21
424 39
6.7 0.3bc

1.37 0.06a
1.49 0.05a
4.15 0.10a
4.04 0.10a
2.94 0.09a
2.87 0.08a
1.20 0.01a
1.18 0.02a
0.41 0.01a
52.8

1.44 0.05ab
1.49 0.03a
3.76 0.15ab
3.85 0.12ab
2.52 0.13b
2.58 0.11ab
1.24 0.02a
1.27 0.02ab
0.49 0.02a
52.6

1.61 0.09bc
1.73 0.10b
3.63 0.01bc
3.61 0.12ab
2.37 0.02b
2.36 0.06bc
1.26 0.03ab
1.26 0.06a
0.53 0.02a
52.1

1.67 0.02c
1.78 0.02b
3.48 0.16bc
3.46 0.16b
2.00 0.10c
1.98 0.11cd
1.48 0.08b
1.47 0.07bc
0.74 0.04b
53.1

1.87 0.04d
1.88 0.05b
3.26 0.14c
3.51 0.23b
1.80 0.12c
1.94 0.20d
1.47 0.13b
1.57 0.12c
0.83 0.11b
50.6

Values as mean standard error, signicant differences between treatments are indicated by different letters in superscript determined by ANOVA, post hoc Students t-test,
n = 15.
f
Sugars determined as soluble solids % w/w by refractive index, sugar per berry was calculated as Brix  0.59  17  berry weight (g) according to Deloire (2011), sugar/
berry/day estimated from net accumulation between harvest dates.
g
Titration end-point to pH of 7 expressed as tartaric acid units.
h
Neutral sugars determined as their 1-phenyl-3-methyl-5-pyrazolone derivatives.
i
Uronic acids determined as galacturonic acid units.
j
Anthocyanin determined colorimetrically at 520 nmin 1 M HCl, expressed as malvidin-3-glucosideunits.
k
Tannin determinedby themethyl-celluloseprecipitation assay expressedin epicatechin units pergberry freshmass orper berry.
l
Determined as [skin tannin]/[seed tannin].
m
EGC, epigallocatechin subunits as a w/w percentage of skin tannin extension subunits determined using phloroglucinolysis.

cold-stabilisation. As expected, malic acid concentration decreased

with grape maturity (Coombe, 1992), whereas acetic acid, citric
acid and tartaric acid showed no signicant change. On the other
hand, trends in succinic acid and glycerol corresponded to that of
ethanol, with higher concentrations in wines from later harvest
dates.
Both total anthocyanin and tannin in wines increased signicantly from H1 to H5, and reected increases in both bisulphiteresistant (non-bleachable) pigments and wine colour density
(Table 2). By comparison with grape composition, anthocyanin
and wine colour density more closely reected changes in grape
anthocyanin concentration during ripening, increasing with each
successive harvest stage (Tables 2 and 3). Total wine tannin concentration, on the other hand, showed a poor relationship with
the trend of decreasing grape tannin (Table 1) from H1 to H5. Interestingly, the proportion of skin tannin (as % of total wine tannin)
increased progressively in wines from the earliest to the latest
harvest. This is noteworthy, in that differences in extractability
between grape-derived skin and seed tannins may exist, which
are not reected in changes in their total extractable levels using
aqueous acetone. Cadot, Caille, Samson, Barbeau, and Cheynier
(2012) have noted a similar trend for Cabernet Franc, whereby
advancing grape maturity was positively correlated with wine tannin and epigallocatechin, and likewise suggested that the extractability of grape tannins is inuenced by ripening.
The increasing proportion of extracted skin tannin was reected
in a signicant increase in wine tannin mDP and molecular mass
by phloroglucinolysis in H5 relative to the other treatments. Conversely, the estimation of tannin molecular mass by GPC showed

that a minor, but signicant decrease occurred from H1 to H5,

rather than the expected increase based on the phloroglucinolysis
results. A detailed analysis of the GPC elution prole of wine tannins puried from the different harvest treatments was undertaken in order to account for these differences in molecular mass
distribution (Fig. 1A). In terms of total polymeric material absorbing at 280 nm isolated from wine, there was a clear increase observed with later harvest. However, it is evident that in the
wines from later harvest dates there was an increase in the proportion of later-eluting, apparently smaller tannins which may
account for the shift in the elution prole to one of a lower average
molecular mass (at 50% elution). The contribution of material
absorbing at 520 nm, i.e. derived from the incorporation of anthocyanins or their derivatives into the tannin polymer, increased progressively from H1 to H5 (Table 2). This 520 nm-absorbing
material was found to be bound to tannin, and did not co-elute
with the later-eluting malvidin-3-glucoside at 1617.5 min
(Fig. 1B). In addition, the contribution of 520 nm-absorbing material was of an apparent lower molecular mass, eluting between
14.5 min and 16 min (Fig. 1B), consistent with the increase in
280 nm absorbance in this elution range for the wines produced
from the later harvest dates. It is therefore unlikely that changes
in the GPC elution prole between 14.5 and 16 min reect a true
shift in tannin polymer size, and may rather be the result of
changes in hydrodynamic volume of the molecule due to anthocyanin incorporation, or possibly other structural modications.
Therefore, the increase in earlier-eluting material from 12 to
13.5 min in H4 and H5 may more accurately reect the increased
proportion of higher mDP tannins derived from grape skin, in

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K. Bindon et al. / Food Chemistry 138 (2013) 16961705

Table 2
Wine compositional analysis. Ethanol concentration, fermentation efciency, acidity and non-volatiles in wines produced at different levels of grape ripeness in 2010 where H1
was the earliest (16th February) and H5 the latest (17th March) harvest date.

Ethanol [% v/v]
Fermentation efciency [g/L per 1% (v/v)]f
Residual sugar [g/L]g
Glycerol [g/L]
pH
Titratable acidity pH 7.0 [g/L]h
Acetic acid [g/L]
Citric acid [g/L]
Malic acid [g/L]
Succinic acid [g/L]
Tartaric acid [g/L]
Neutral polysaccharide [mg/L]i
Acidic polysaccharide [mg/L]i
Total polyaccharide [mg/L]i
Anthocyanin [mg/L]j
SO2-resistant pigments [a.u.]k
Wine colour density [a.u.]k
Total tannin [mg/L]l
Skin tannin [%]m
Tannin mDPm
Tannin molecular mass (g/mol by subunit)m
Tannin molecular mass (g/mol by GPC)n
Tannin 520/280 ratio (%)n

H1

H2

H3

H4

H5

11.77 0.03e
17.03 0.27a
0.40 0.00b
6.97 0.03d
3.36 0.02d
6.30 0.12a
0.27 0.09a
0.30 0.00a
3.03 0.03a
1.33 0.03d
1.90 0.00abc
84.7 6.2a
137.7 42.6a
222.4 37.1a
411 22a
1.04 0.08a
7.64 0.17a
731 61ab
56.8 0.52a
7.15 0.10ab
2148 30a
3315 75a
6.77 0.24a

12.87 0.09d
17.00 0.15a
0.40 0.06b
7.63 0.07c
3.50 0.03b
5.70 0.00bc
0.20 0.06a
0.33 0.03a
2.73 0.07b
1.50 0.00c
1.70 0.00c
85.0 3.8a
94.5 5.8ab
179.4 3.9ab
529 11b
1.30 0.05ab
9.95 0.26b
750 38ab
58.8 0.57a
7.04 019a
2114 58a
3073 122ab
7.96 0.15b

13.57 0.09c
16.95 0.30a
0.53 0.09b
8.13 0.03b
3.41 0.02cd
6.00 0.15ab
0.27 0.03a
0.33 0.03a
2.40 0.06c
1.60 0.00bc
2.07 0.09a
91.5 14.8ab
77.5 12.8abc
169.0 2.3ab
592 23b
1.82 0.30c
11.34 0.31c
638 8a
63.5 0.82b
7.72 0.06c
2317 18b
2987 6ab
8.35 0.12bc

14.2 0.10b
16.64 0.30a
0.40 0.06b
8.27 0.14b
3.62 0.01a
5.37 0.07c
0.30 0.01a
0.30 0.00a
2.37 0.09c
1.73 0.09ab
1.80 0.12bc
121.3 18.0b
31.6 5.9bc
152.9 22.8b
657 1.78c
1.74 0.10bc
11.26 0.17c
786 46b
64.1 0.19b
7.55 0.26bc
2264 80ab
2914 207b
8.73 0.22c

15.50 0.06a
16.63 0.47a
0.90 0.00a
9.47 0.03a
3.46 0.03bc
6.33 0.13a
0.37 0.03a
0.30 0.00a
2.27 0.03c
1.87 0.03a
2.03 0.09ab
114.6 4.2ab
25.8 7.9c
140.4 4.0b
728 30d
2.59 0.05d
14.51 0.21d
1088 43c
71.8 0.89c
8.39 0.07d
2519 23c
2934 30b
9.61 0.13d

Values as mean standard error, signicant differences between treatments are indicated by different letters in superscript determined by ANOVA, post hoc Students t-test,
n = 15.
f
Fermentation efciency is the concentration of sugar needed to produce 1% (v/v) of ethanol.
g
Residual sugar determined as glucose and fructose by HPLC.
h
Titration end-point to pH of 7 expressed as tartaric acid units.
i
Polysaccharide calculated as the sum of neutral sugars from their corresponding alditol acetates determined by GCMS and uronic acids determined colorimetrically as
galacturonic acid units.
j
Anthocyanin as malvidin-3-glucoside units determined colorimetrically.
k
a.u., absorbance units, wine colour density corrected for SO2 in the presence of acetaldehyde.
l
Tannin determined by the methyl-cellulose precipitation assay expressed in epicatechin units.
m
Tannin compositional information determined by phloroglucinolysis: skintannin calculated fromtheratio of epigallocatechin in extension subunits in grape skin tannin
(Table 1)as aproportion of wine tannin extension subunits, mDP, mean degree of polymerisation.
n
Determined by gel permeation chromatography (GPC), molecular mass average at 50% elution, absorbance ratio calculated from 280 and 520 nm peak areas of the tannin
isolate.

Table 3
Compositional analysis of neutral monosaccharide composition as mol% in polysaccharides isolated from wines produced at different levels of grape ripeness in 2010 where H1
was the earliest (16th February) and H5 the latest (17th March) harvest date.
H1
Rhamnose
Fucose
Arabinose
Xylose
Mannose
Galactose
Glucose

H2
a

9.98 0.27
0.62 0.04ab
24.34 0.33a
0.91 0.05a
28.94 0.47a
27.22 0.29abc
8.09 0.11a

H3
a

9.98 0.46
0.69 0.04a
24.72 0.78a
0.88 0.08ab
28.84 0.90bc
27.03 0.24bc
7.86 0.51a

H4
a

9.90 0.51
0.63 0.04a
23.73 0.72a
0.88 0.06ab
31.14 0.97ab
26.44 0.64c
7.27 0.25a

H5
b

5.24 0.63
0.42 0.10bc
18.90 1.08b
0.68 0.10bc
41.18 1.32c
28.46 1.14ab
5.11 0.37b

4.23 1.39b
0.27 0.07c
17.84 1.07b
0.60 0.03c
43.93 2.32a
29.08 0.29a
4.04 0.03c

Values as mean standard error, signicant differences between treatments are indicated by different letters in superscript determined by ANOVA, post hoc Students t-test,
n = 15.

accordance with the observations using phloroglucinolysis. This

nding conrms observations of Cadot et al. (2012) who noted a
decrease in thiolysis yield in tannins from Cabernet Franc wines
produced from grapes of more advanced ripeness. They suggested
that this could reect enhanced formation of thiolysis-resistant
anthocyanin-tannin adducts in wines made from riper grapes,
associated with increasing levels of anthocyanin.
Total wine-soluble polysaccharides decreased by harvest date
only when H1 was compared with the other treatments. However,
there were clear compositional changes, notably a loss in the acidic
polysaccharide fraction and increases in the neutral polysaccharide
fraction in wines from later harvest dates (Table 2). Analysis of
neutral monosaccharide composition revealed that trends from

H1 to H5 were a proportional loss in arabinose, with signicant increases in mannose (by mol%) (Table 3). Trends in the proportional
composition of the minor sugars, rhamnose, fucose, xylose and glucose also decreased in wines from later harvest dates, but galactose
stayed relatively constant. Since the observed trends were consistent with a loss in grape-derived galacturonans through vinication, and increases in yeast-derived polysaccharides (i.e., MPs)
with later harvest date, this was further assessed by size exclusion
chromatography (Fig. 2). Wines from the earlier harvest dates had
a higher proportion of the lower molecular mass polysaccharides,
which correspond to the elution of RGs, consistent with the observation of a higher acidic polysaccharide component and rhamnose.
Wines produced from advanced grape maturity stages showed a

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K. Bindon et al. / Food Chemistry 138 (2013) 16961705

Fig. 2. Elution prole determined by size exclusion chromatography of mannoproteins (MP), arabinogalactan proteins (AGP) and rhamnogalacturonan (RG) polysaccharides from wines produced from early (H1, 12% v/v ethanol) and late (H5,
15.5% v/v ethanol) harvested grapes.

Fig. 1. Gel permeation chromatography analysis of (A) elution proles at 280 nm of

wine tannins from different harvest dates where H1, earliest harvest, 12% v/v
ethanol and H5, latest harvest, 15.5% v/v ethanol and (B) contribution of wine
tannin peak areas at 280 nm and 520 nm by comparison with monomeric malvidin3-glucoside (malvidin-3-glucoside had the same elution prole at both 280 nm and
520 nm).

proportional loss of late-eluting, low molecular mass polysaccharide (galacturonans), and increased higher high molecular mass
material, consistent with the enrichment of the MP fraction. The
increased concentration of MPs may be due to enhanced yeast
metabolism in higher-ethanol wines, and may also have been associated with increased secretion of endo-polygalacturonase (PGU1),
an enzyme which can be expressed in the S. cerevisiae PDM strain
used (EC1118) (Novo et al., 2009; Radoi, Kishida, & Kawasaki
2005). Since polysaccharide quantity and composition did not differ between harvest dates in juices, the loss in acidic galacturonans
from later-harvest wines may potentially be associated with an increased PGU1 activity.
A further important consideration is the role of inter-molecular
interactions between wine tannins and polysaccharides. A pertinent study by Riou, Vernhet, Doco, and Moutounet (2002) observed the behaviour of wine polysaccharide classes in either
enhancing or restricting the aggregation of seed tannins in model
solution. High molecular weight AGP and MPs were shown to reduce the particle size of tannin aggregates, maintaining tannin solubility in colloid complexes. Conversely, an RG dimer
(rhamnogalacturonan II) enhanced tannin aggregation, thereby
potentially enhancing tannin precipitation. In the current study,

the higher tannin concentration in later-harvest wines could lead

to an increased potential for aggregate formation, but a higher
MP concentration could favour the maintenance of tannin in colloid complexes. Since juice-soluble polysaccharides (Table 1) were
both higher in the concentration and proportion of acidic polysaccharide residues than those in wine (Table 2), the potential exists
that some loss of tannin could have occurred via aggregation and
precipitation as co-aggregates with soluble galacturonan-rich
polysaccharide. While this would need to be veried experimentally for wine tannin, this may offer a reasonable explanation for
the lack of RGs in later-harvest wines.
An additional, anecdotal observation in the winemaking process
was that treatments H4 and H5 had reduced lterability. It has
been observed that the presence of high molecular mass MPs reduces wine permeation ux (Vernhet, Pellerin, Belleville, Planque,
& Moutounet, 1999), and the potential exists that some grape-derived polysaccharides present at the end of ferment were removed
from the nished wines due to fouling of the membrane with MPs.
Since the presence of a higher pectic polysaccharide to MP ratio has
also been noted to enhance wine permeation ux (Vernhet et al.,
1999), it is possible that this contributed to polysaccharide recovery differences between H1 to H3 and the wines produced from the
later-harvested grape sources.
3.4. Wine volatiles
All volatile compounds detected in more than one wine are
presented in Table 4. b-Damascenone and b-ionone were the only
C13-norisoprenoids detected in wines, b-damascenone showed no
signicant differences by harvest date, whereas b-ionone was only
detected in H1 wines at 0.5 lg/L (data not shown). The monoterpene linalool showed no signicant differences between harvest
treatments, and in Cabernet Sauvignon this compound has been
found to be synthesised de novo by yeast metabolism, independently of grape-derived precursors available during fermentation
(Keyzers & Boss, 2010). The concentration of IBMP, the only pyrazine detected in wines, decreased signicantly by harvest date
from H1 to H5. This decrease was a more signicant trend in wine
than that observed for IBMP extracted from grapes (Table 1). The
extracted levels of IBMP in wines were higher than the detectable
levels in grapes. This may be consistent with an incomplete extraction from the grape solids in our analysis of IBMP in grape juice. To
better approximate wine IBMP using grape measurements, Ryona,

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K. Bindon et al. / Food Chemistry 138 (2013) 16961705

Table 4
Analysis of volatile composition of wines produced at different levels of grape ripeness in 2010 where H1 was the earliest (16th February, 12% v/v ethanol) and H5 the latest (17th
March, 15.5% v/v ethanol) harvest date.
H1

H2

H3

10.3 0.35b
3.0 0.00a
14.3 0.4a

10.5 0.00b
2.7 0.35a
11.7 0.4b

11.7 0.17a
2.7 0.35a
8.7 0.4c

9.8 0.17b
2.3 0.35a
8.0 0.6cd

11.8 0.58a
3.0 0.00a
7.0 0.6d

2.0 0.00d
nd

3.0 0.00c
nd

3.3 0.35c
nd

4.0 0.00b
1.7 1.67b

5.3 0.35a
10.7 0.35a

Esters
Ethyl acetate [mg/L]
Hexyl acetate [mg/L]
2-Methylbutyl acetate [mg/L]
3-Methylbutyl acetate [mg/L]
2-Phenylethyl acetate [mg/L]
Ethyl propanoate [mg/L]
Ethyl 2-methylpropanoate [mg/L]
Ethyl butanoate [mg/L]
Ethyl 3-methylbutanoate [mg/L]
Ethyl hexanoate [mg/L]
Total ethyl esters [mg/L]f
Total acetate esters [mg/L]f
Total esters [mg/L]f

28.9 1.44d
77 6.35a
90 9.2d
0.9 0.12d
70 11b
248 14c
47.3 4.21bc
301 18c
14.4 0.75c
1.0 0.12d
1.6 0.12d
1.1 0.12c
2.7 0.23d

36.1 1.91c
63 4.62b
119 10cd
1.0 0.12cd
68 1.73b
284 13c
41.3 0.64c
326 23c
13.8 0.06c
1.0 0.06cd
1.7 0.06cd
1.3 0.12bc
3.0 0.17cd

44.1 3.23b
64 2.89ab
138 13bc
1.3 0.12b
114 35ab
335 8.7b
56.3 5.20b
386 5.77b
21.8 2.71b
1.3 0.06ab
2.1 0.06b
1.6 0.12b
3.7 0.17b

45.2 0.23b
67 1.73ab
175 13b
1.3 0.00bc
85 13ab
357 0.6b
42.2 0.75c
348 2.89bc
14.4 0.40c
1.2 0.00bc
1.9 0.00bc
1.7 0.00b
3.6 0.00bc

55 0.12a
57 2.89b
235 19a
1.8 0.06a
165 43a
419 19a
68.2 1.79a
474 19a
27.3 1.15a
1.4 0.00a
2.4 0.06a
2.2 0.12a
4.7 0.12a

Alcohols
Hexanol [mg/L]
Z-3-Hexen-1-ol [mg/L]
Butanol [mg/L]
2-Methylpropanol [mg/L]
2-Methylbutanol [mg/L]
3-Methylbutanol [mg/L]
Total higher alcohols [mg/L]

5.6 0.12a
27.3 0.87a
490 17d
21.7 0.69b
68 0.58b
179 2.89a
269 2.31a

4.7 0.17b
18.7 0.35b
648 26c
24.1 1.85ab
79 0.58ab
185 1.15a
288 2.31a

4.4 0.12b
13.0 0.75c
778 23b
23.6 0.52ab
77 8.08ab
187 22a
288 30a

4.4 0.00b
9.3 0.23d
843 13b
24.5 0.17ab
89 4.62a
205 9.2a
320 14a

3.0 0.17c
6.1 0.12e
1275 21a
25.7 0.98a
88 4.04a
205 11a
320 16a

Isoprenoids and pyrazines

b-Damascenone [mg/L]
Linalool [mg/L]
Isobutyl methoxypyrazine [ng/L]
Thiols
Dimethyl sulde [mg/L]
Methyl thioacetate [mg/L]

H4

H5

Values as mean standard error, signicant differences between treatments are indicated by different letters in superscript determined by ANOVA, post hoc Students t-test,
n = 15; nd, not detected.
f
Values exclude ethyl acetate.

Pan, and Sacks (2009) have observed that IBMP extraction into
grape juice from solids may be facilitated by the application of
higher extraction temperature and extended skin contact time, giving a better approximation of the levels expected in wine.
Hexanol and (Z)-3-hexen-1-ol, and hexyl acetate, all C6 compounds originating from grape-derived precursors, also showed
lower concentrations in wines with advancing harvest date. The
C6 alcohols are thought to be derived from C18 fatty acids via the
lipoxygenase pathway and alcohol dehydrogenase, either in situ
during grape ripening, or under the oxidative conditions present
when fruit is crushed (Dennis et al., 2012; Kalua & Boss, 2009).
Hexyl acetate, on the other hand, has been found to be at low levels, or absent in grapes, and it appears that alcohol acetyl transferase activity within Cabernet Sauvignon favours the formation of
(Z)-3-hexenyl acetate as the principal biosynthetic pathway (Kalua
& Boss, 2009). However, (Z)-3-hexenyl acetate was absent in the
wines of the current study. Recently, a direct relationship between
hexanol concentration in ferment, and hexyl acetate production in
wine via the action of yeast alcohol acetyl transferase has been
demonstrated (Dennis et al., 2012). This most likely accounts for
the observed decline in hexanol and hexyl acetate in wines produced from grapes at later maturity stages.
Apart from hexyl acetate, the total concentration of all esters,
excluding ethyl acetate, increased in wines from H1 to H5. This increase was caused by changes in both ethyl esters and acetate esters (Table 4). Formation of esters depends on both the
concentration of their precursors and yeast metabolism (Saerens
et al., 2008; Verstrepen et al., 2003). Wines produced from grapes
of advancing maturity resulted in higher concentrations of most
esters, namely ethyl acetate, ethyl propanoate, ethyl butanoate,
2-methylbutyl acetate and 3-methylbutyl acetate. Although ethyl

2-methylpropanoate, ethyl 3-methylbutanoate, ethyl hexanoate

and 2-phenylethyl acetate did not show a clear trend with grape
maturity, they exhibited higher concentrations at H5 than at H1.
The concentration of the yeast-derived low molecular weight volatiles dimethyl sulde and methyl thioacetate in wines were very
low, but also increased from H1 to H5, consistent with a higher
general yeast metabolism in musts of higher starting sugar
content.
Butanol was the only higher alcohol which showed a linear increase in concentration in wines with advancing harvest date. 2Methylpropanol, 2-methylbutanol and 3-methylbutanol showed
increased concentration from H1 to H2 and no signicant differences thereafter (Table 4).
3.5. Response of yeast metabolism to increases in must sugar content
Increasing must sugar concentration as a result of advanced
grape maturity, resulted in signicant effects on yeast metabolism.
As the production of ethanol by yeast increased, there were concomitant increases in most yeast-derived metabolites, including
low molecular weight sulphur-containing volatiles, esters and
higher alcohols. We have also previously discussed the increased
secretion of yeast-derived mannoproteins in higher-ethanol wines,
with the corresponding loss of polysaccharides rich in uronic acids
and rhamnose, potentially the result of enhanced yeast polygalacturonase activity.
However, it is noteworthy that the acids acetic acid, citric acid
and malic acid showed a different prole. Since the carbon fraction
from sugar ending up as acetic acid or citric acid is very small (less
than 0.15%), the relatively low resolution of the quantication
method at low concentrations could explain why these acids were

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K. Bindon et al. / Food Chemistry 138 (2013) 16961705

apparently unaffected by higher sugar concentration. Malic acid is

well known to decline in ripening grapes (Coombe, 1992), and this
factor alone may account for the observed decline in grape juice pH
and TA by harvest stage (Table 2), although malic acid concentration in must was not analysed in this study. Since the wines did
not undergo malolactic fermentation, decreases in wine malic acid
concentration with advancing ripeness stage may be the direct result of its metabolism as grape ripening progressed. As S. cerevisiae
PDM has a limited activity for malic acid degradation during fermentation (Redzepovic et al., 2003), increased yeast metabolism
could partially explain lower concentrations of malic acid in higher
alcohol wines.
4. Conclusions
The current study has demonstrated signicant changes in wine
matrix chemistry which are inuenced primarily by grape maturity, and secondarily by yeast metabolism in response to changes
in must sugar concentration. Many of these effects on wine chemistry in turn have implications for wine sensory properties, and the
challenge within this study is that there are multiple interactions
which may impinge to induce a sensory response. The research
has also included a detailed sensory analysis, which will be presented as a follow-up manuscript. Nevertheless, we note some
key observations from this research that may signicantly impinge
on wine style:
a. Increases in various yeast-derived volatiles in response to
higher must sugar concentration, may indicate that a relationship between sugar concentration and yeast metabolism
exists, and warrants further investigation. The initial must
FANs were low, and therefore was between 9% and 21% of
the nitrogen available during ferment, but it cannot be
excluded that differences in yeast metabolism may have
been driven by minor differences in FAN amino acid composition. While this result is not unexpected, the notable effect
of yeast metabolism on concentrations of fermentationderived esters is likely to signicantly impact upon the perception and attributes of fruity aroma and avour in wines,
as many of the esters which are recognised as being of signicant odour impact were above their detection threshold
(Escudero et al., 2007; Sumby, Grbin, & Jiranek, 2010).
b. The decrease in IBMP and volatiles derived from C6 precursors in wines made from grapes at advancing stages of maturity suggests an independence from yeast metabolic
turnover. The analysis of IBMP in Cabernet Sauvignon is
well-known to decline with grape ripening, but from this
perspective C6 compounds have received less attention in
research, in particular their formation in wine. Kalua and
Boss (2009) showed variability in the trends of either
increase or decline of C6 volatiles in grapes with ripening.
Later work by this group conrmed the dependence of C6
volatiles in wines on the concentration of lipoxygenase precursors pre-ferment (Dennis et al., 2012). We note that the
analysis of C6 precursors may serve as valuable markers for
grape ripeness in this variety, alongside IBMP. In addition
to the assay of in situ grape C6 precursors (Kalua & Boss,
2009), a valuable comparison would be an assay of the de
novo generation of C6 volatiles from fatty acid precursors
via the lipoxygenase pathway, under oxidative conditions
which mimic fruit crushing.
c. A shift was observed whereby increased skin tannin contribution, and reduced seed tannin was found in higher alcohol
wines, concomitant with higher total wine tannin, colour,
anthocyanin and bisulphite-resistant pigments. The role that

biochemical changes induced by grape ripening might play

in affecting tannin extractability is poorly understood.
Research in this area has been limited by the choice of
extraction method, and we have noted in the current work
that the use of a 70% v/v acetone:water solvent provided a
poor estimate of wine-soluble tannin with grape maturity.
However, the use of a 70% v/v acetone:water extract has
enabled the demonstration of a strong correlation of grape
skin tannin concentration with wine tannin concentration,
but a poor correlation for the respective concentrations of
seed and wine tannin (Ristic et al., 2010). We therefore propose that skin and seed tannin extractability are differentially affected by ripening, and that further optimisation of
the analytical methodology used is required in order to accurately model these effects in a grape to wine scenario.
d. The enrichment of yeast-derived MPs and reduced grapederived galacturonans in higher alcohol wines may be consistent with enhanced yeast metabolism in musts of higher
sugar concentration, but we note that the loss of galacturonans corresponded to both higher wine tannin and increased
MPs. Since MPs may protect the loss of tannin by limiting
tannin aggregation, and galacturonans may promote tannin
aggregation (Riou et al., 2002), selectivity in tannin-polysaccharide interactions may have contributed to the observed
shifts in polysaccharide prole. This highlights a greater
need to understand the contribution of tannin-polysaccharide interactions in promoting the formation of either colloids or aggregates, in particular for a more complex
matrix, such as wine which contains both diverse polysaccharide classes and variable tannin concentrations.

Acknowledgements
The authors would like to thank Pernod Ricard Australia (Orlando-Wyndham Vineyards) for the donation of grape samples. We
particularly thank Dr. Mike McCarthy of the South Australian Research and Development Institute (SARDI) for access to eld trial
data. We would like to thank the ARC Centre of Excellence in Plant
Cell Walls (University of Melbourne) for the use of their facilities
and for technical advice. We would like to thank AWRI staff for
their contributions to winemaking and sample analysis: Gemma
West, Stella Kassara, Fiona Brooks, Dimitra Capone, Natoiya Lloyd,
Richard Gawel, the AWRI Commercial Services laboratory and the
AWRI Trace Laboratory. The Australian Wine Research Institute, a
member of the Wine Innovation Cluster in Adelaide, is supported
by Australian grape growers and winemakers through their investment body, the Grape and Wine Research and Development Corporation, with matching funds from the Australian Government.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2012.
09.146.
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